Cytisine is neuroprotective in female but not male 6-hydroxydopamine lesioned parkinsonian mice and acts in combination with 17-β-estradiol to inhibit apoptotic endoplasmic reticulum stress in dopaminergic neurons

2.1 Mice

All experiments were conducted in accordance with Texas A&M University IACUC regulations and protocols (protocol # 2019-0346) and were not pre-registered. Non-gonadectomized wild-type C57BL/6 female mice, and wild-type C57BL/6 male mice were used in behavioral studies. All mice were obtained from Taconic (Taconic), and were 8 weeks old at the start of behavioral studies. Food and water were provided ad libitum. Mice were housed with littermates in ventilated cages and maintained on a 12 hr light–dark cycle. Timed pregnant mice for generating dopaminergic (DA) cultures were obtained from the Texas A&M Institute for Genomic Medicine (TIGM). All survival surgeries were conducted with continuous isoflurane anesthesia. Routine surgical procedures included application of eye lubricant, and administration of i.p. saline following surgeries. Pain and suffering was minimized in 6-OHDA-treated animals by providing moistened peanut butter mush kept on the cage floor to prevent weight loss following lesions. We did not administer analgesics or antibiotics to the mice in order to avoid the possibility of drug-drug interactions, which may alter the pharmacokinetics of cytisine. For generating DA cultures, timed pregnant mice were asphyxiated with carbon dioxide within 2–3 min and a secondary euthanasia with cervical dislocation was performed prior to extracting embryos. No randomization was performed to allocate subjects in the study.

2.2 Procedure for striatal injections of 6-OHDA in mice

6-OHDA was stereotaxically injected into the mouse dorsolateral striatum (DLS) using previously described methods (Srinivasan et al., 2015; Srinivasan, Lu, et al., 2016). A 5 mg/ml stock solution of 6-OHDA (Sigma, St. Louis, MO) was prepared in 0.9% saline with 0.2% ascorbic acid and frozen at −80°C until use. Two microliters of the stock solution were withdrawn into a beveled glass injection pipette using a motorized Pump 11 Pico Plus Elite pump (Harvard Apparatus), attached to a stereotaxic frame (Kopf Instruments). Mice were anesthetized using isoflurane dispensed through a SomnoSuite Low Flow Anesthesia System (Kent Scientific). 6-OHDA was unilaterally injected into the DLS at a rate of 750 nl/min. Coordinates for 6-OHDA injections into the DLS were 0.8 mm anterior to bregma, 2.0 mm lateral to midline, and 2.4 mm ventral to the pial surface.

2.3 Protocol for in vivo mouse experiments

Figure 1a shows the protocol for in vivo experiments in mice. Starting one week prior to 6-OHDA treatment (day −7), female and male mice were injected intraperitoneally (i.p), on alternate days, with 200 µl of either 0.9% normal saline or 0.2 mg/kg -(-) cytisine (Sigma), dissolved in 0.9% normal saline. Mice were weighed on alternate days, just prior to i.p. cytisine or saline injections and over the course of the entire experiment. Behavioral assays were performed on days −7 (baseline), 7, 14, and 21 where day 0 is the time point for 6-OHDA injection. After testing on day 21 all mice were deeply anesthetized with isoflurane and perfused with 10% formalin. Following perfusion, midbrain sections were collected for quantification of SNc DA neurons after tyrosine hydroxylase (TH) immunostaining.

2.4 Apomorphine rotations

Each mouse was placed in a 5-gallon circular bucket (33 cm diameter), and allowed to acclimate for at least 15 min prior to testing. For testing, apomorphine (0.5 mg/kg in 0.9% saline; Sigma, cat # 1041008) was injected i.p and immediately afterward, contralateral rotational behavior was video recorded for 15 min with a ceiling mounted camcorder (Movies S1, S2). The total number of contralateral rotations made during this 15-min test was counted using Ethovision XT (Noldus, RRID:SCR_000441). All detection settings on Ethovision were maintained across cohorts of mice and experiment days.

2.5 Ladder rung walking test and analyses

A custom-made apparatus with 0.5-cm-thick plexiglass sidewalls encasing 33 metal dowel rungs (0.5 cm diameter, evenly spaced at 1.5 cm intervals), was used for the ladder rung walking test (Figure 2f and Movies S3, S4). Mice were video recorded while walking across the ladder rungs, freely in both directions, for 2.5 min. Video recordings were analyzed post hoc for gait patterns and footslips.

For gait pattern analysis, an alphabetical value was assigned to each paw: right forepaw = a, right hindpaw = b, left forepaw = c, left hindpaw = d. A normal gait pattern was identified as movement of a forepaw (a or c) followed by movement of the contralateral hindpaw (b or d) or movement of a hindpaw (b or d) followed by the contralateral forepaw (a or c). Thus, examples of normal gait patterns are a-d-c-b or c-b-a-d (Movie S3), whereas abnormal gait patterns are a-c-b-d or c-a-d-b (Movie S4) (Figure 2d). Alphabetical scoring of gait patterns was used to determine the percent times that mice displayed normal versus abnormal gait on days −7, 7, 14 and 21.

For foot slip analysis, the number of forepaw foot slips were manually scored when the mouse was walking in a straight direction (Figure 2f). Foot slips that occurred during behaviors such as turning, rearing, or when the mouse was stationary were not counted. The foot slip data shown in Figure 2g are the fold change from baseline foot slips observed on day −7.

2.6 Long-term primary DA-astrocyte co-cultures

Each independent primary mouse DA astrocyte co-culture consisted of pooled midbrain cells derived from mouse embryos of four to seven timed pregnant mice at embryonic day 14. Dissociated midbrain cells from each pooled culture were plated on 8 to 10 #1 12 mm diameter glass coverslips. To obtain DA-astrocyte co-cultures, the ventral midbrain was dissected from mouse embryos as previously reported (Bancroft & Srinivasan, 2020; Henley et al., 2017; Srinivasan, Henley, et al., 2016). Following dissection, cells were dissociated using papain (Worthington Biochemical Corporation, cat # LS003126), DNase treatment (Sigma, cat # DN25), and mechanical trituration. 200,000 cells were plated on the coverslips coated with poly-L-lysine (Sigma, cat # P4832), poly-l-ornithine (Sigma, cat # P4957), and laminin (Sigma, cat # L2020). Cells were maintained in culture with neurobasal medium (ThermoFisher, cat # 21103049) supplemented with B-27 supplement (ThermoFisher, cat # 17504044), 5% heat-inactivated horse serum (ThermoFisher, cat # 26050088), GlutaMAX (ThermoFisher, cat # 35050061), 0.2% L-ascorbic acid (Sigma, cat # A7506), 10,000 U/mL penicillin-streptomycin (ThermoFisher, cat # 15140122), 60 mg/ml kanamycin (Sigma, cat # 60615), and 125 mg/ml ampicillin (Sigma, cat # A9393). Primary DA neuron astrocyte co-cultures were maintained for a total of 4 weeks and the culture medium was exchanged every 2–3 days. For ER stress marker experiments, cultures were treated with 200 nM cytisine (Sigma, cat # 5052270001) dissolved in phosphate-buffered saline (PBS) for 2 weeks starting on day in vitro 14 (d.i.v 14). On d.i.v 26, experimental cultures were treated with 400 µM 6-OHDA dissolved in PBS containing 0.2% ascorbic acid (Sigma, cat # H4381), 400 µM 6-OHDA for 48 hr + 200 nM cytisine for 2 weeks or 10 μM doxorubicin for 48 hr (Sigma, cat # D1515). For 17-β-estradiol experiments, cultures were treated with 10 nM 17-β-estradiol, dissolved in 100% ethanol (Sigma, cat # E2758) + 200 nM cytisine for 2 weeks and on d.i.v 26, some cultures were treated with 400 µM 6-OHDA for 48 hr. On average, our 4-week-old long-term primary DA-astrocyte co-cultures yielded 20%–30% TH positive neurons.

2.7 Immunostaining

Antibodies for Sec24D and ER stress markers (ATF6, XBP1, CHOP) have been previously validated (Srinivasan, Henley, et al., 2016). For staining DA cultures, cells were fixed in 10% formalin (VWR, cat # CA71007-348) for 20 min at RT, rinsed in PBS and permeabilized with 0.01% Triton X-100 for 1 min at RT. Cultures were blocked with 10% normal goat serum (NGS) (Abcam, cat # ab7481, RRID:AB_2716553 for 40 min at RT and incubated with the appropriate primary antibody in 1% NGS at 4°C, overnight. Primary antibodies were chicken anti-TH (1:1,500; Abcam, cat # ab76442, RRID:AB_1524535), rabbit anti-CHOP (1:500; Abcam, cat # ab10444, RRID:AB_2245733), rabbit anti-ATF6 (1:500; Abcam, cat # ab203119, RRID:AB_2650448), rabbit anti-XBP1 (1:500; Abcam, cat # ab37152, RRID:AB_778939), rabbit anti-Sec24D (1:500; ThermoFisher, cat # PA5-65793, RRID:AB_2662256), and rabbit anti-cleaved caspase-3 (1:500; Cell Signaling, cat # 9661S, RRID:2341188). Cells were then incubated with the appropriate secondary antibody in 1% NGS for 1 hr at RT with constant agitation. The secondary antibodies were goat anti-chicken Alexa Fluor 594 (1:2000; Abcam, cat # ab150176, RRID:AB_2716250) and goat anti-rabbit Alexa Fluor 488 (1:1,000; Abcam, cat # ab150077, RRID:AB_2630356). Staining for each protein was performed sequentially to prevent cross reactivity. To detect ER stress markers ATF6, XBP1, and CHOP, cultures were first labeled with primary and secondary antibodies for the stress markers, followed by TH primary and secondary antibodies. For Sec24D-ERES staining, cultures were first labeled with TH primary and secondary antibodies followed by Sec24D primary and secondary antibodies.

For immunostaining brain sections, mice were deeply anesthetized with isoflurane and transcardially perfused with 1X PBS followed by 10% formalin. Brains were extracted and stored overnight at 4°C in 10% formalin, then dehydrated for 48 hr in 30% sucrose (Sigma, cat # S7903). Midbrain sections (40 μm) were obtained (Microm HM 550 cryostat, ThermoFisher) and stored in 0.01% sodium azide (Sigma, cat # S2002) at 4°C until immunostaining. Following 3× washes with PBS, sections were permeabilized and blocked with 0.5% Triton X-100 + 10% normal goat serum for 1 hr at RT. Sections were co-labeled with rabbit anti-CHOP (1:500; Abcam, cat # ab10444, RRID:AB_2245733) and a chicken anti-TH primary antibody (1:1,500, cat # ab76442) overnight at 4°C followed by a goat anti-rabbit Alexa Fluor 488 (1:1,000, Abcam, cat # ab150077, RRID:AB_2630356) and goat anti-chicken Alexa Fluor 594 secondary antibody (1:2000, cat # ab150176, RRID:AB_2716250) for 1 hr at RT.

2.8 Confocal imaging

Imaging was performed on an inverted Olympus FV1200 confocal laser scanning microscope (Olympus) equipped with a 10x air objective (NA 0.40), a 60× oil immersion objective (NA 1.35) and 488 and 594 nm laser lines. Microscope settings were optimized to obtain non-saturated images, and the same settings for laser power, high voltage, gain, offset, and aperture diameter were maintained across all imaging sessions. For imaging ER stress markers (ATF6, XBP1, and CHOP), images of individual TH⁺ cell bodies were acquired at 60× magnification and a digital zoom of 5–8×. DA neurons were individually imaged utilizing TH fluorescence as a guide to focus on an optical plane with the nucleus sharply demarcated. Thirty to 40 individual TH⁺ neurons from each coverslip were imaged. For Sec24D ERES imaging, z-stacks of individual TH⁺ neurons were obtained with a 0.45 µm step size, and 30–40 TH⁺ cells for each condition were imaged. Average data for each condition (Figures 4-7) were obtained from 60 to 200 TH⁺ neurons, derived from three to four independent long-term DA cultures.

To quantify neuroprotection of the SNc after in vivo behavioral testing, TH⁺ SNc and ventral tegmental area montages were acquired from six midbrain sections per mouse. Montages were stitched from individual images obtained at 10x magnification, spanning the lesioned and unlesioned hemispheres (Figure 3a). Images for each montage were acquired at a single optical plane. The laser power, high voltage, gain, offset, and aperture diameter were adjusted so that pixels were not saturated and imaging parameters were maintained across all sections and mice.

2.9 Image analysis

All image analysis was performed using ImageJ software. To analyze Sec24D-ERES, a z projection was generated for each TH⁺ neuron and Sec24D-labeled ERES structures were manually thresholded. Sec24D-ERES regions of interest (ROIs) were demarcated using the Analyze Particles feature in ImageJ with a ROI area constraint of 0.01–1,000 µm². Sec24D-ERES ROIs were used to extract the number, area, and integrated fluorescence intensity, which is the sum of fluorescence values from all pixels within an ROI. For each experimental condition, 60–150 individual TH⁺ cells from two to four independent mouse midbrain cultures were analyzed for ER stress markers (ATF6, XBP1 or CHOP).

The absence of TH labeling in the nucleus enabled a clear differentiation between cytosolic and nuclear compartments. ROIs were manually drawn around the nucleus and the whole-cell boundary for each TH⁺ neuron, and the mean intensity of ATF6, XBP1, or CHOP was quantified separately for each demarcated region (Figure 5b). To assess percent nuclear translocation of CHOP in SNc DA neurons from immunostained midbrain sections of female and male mice, images of individual TH⁺ SNc DA neurons from the 6-OHDA lesioned side of 40 μm mouse midbrain sections were acquired using a 60× oil immersion objective and 3× digital zoom. A ratio of integrated CHOP fluorescence intensity in the nucleus to the whole cell (nucleus + cytoplasm) was obtained, and percent nuclear CHOP translocation was extracted from this ratio for each DA neuron in the lesioned SNc.

To quantify TH fluorescence in midbrain sections, 10× midbrain montages were thresholded to isolate TH⁺ cell bodies and processes. ROIs from the thresholded images were utilized to obtain integrated fluorescence intensities for the unlesioned and the 6-OHDA lesioned side. Ratios of integrated fluorescence intensity of the 6-OHDA lesioned versus the unlesioned SNc were compared to assess the extent of neuroprotection by cytisine in female and male mice. Ratios of fluorescence intensity were used instead of raw fluorescence intensity to reduce variability across midbrain sections and mice that can occur because of immunostaining artifacts.

2.10 Sampling and statistics

All statistics except for behavioral experiments were performed using Origin 2019 (OriginLab, RRID:SCR_014212) and R programming language (https://www.r-project.org/, RRID:SCR_001905). Statistics for behavioral experiments were performed using Statistical Package for the Social Sciences (SPSS version 18; IBM, RRID:SCR_019096). Experiments were blinded since the analysis was performed by a person other than the experimenter. As a pre-determined exclusion criterion, mice that died prior to the completion of the 28 day in vivo experimental protocol were excluded from analysis. No data points were excluded as outliers. All experiments were conducted during the day, between 9 a.m. to 6 p.m.

The mice reported in this study were tested in separate behavioral experiments across multiple cohorts. We used a total of 22 female and 23 male mice with a ~20% mortality rate after 6-OHDA lesion. Mice that died before the final day of the experiment were not used for weight measurements, behavioral assays, TH intensity, or CHOP intensity. Numbers of mice for weight measurements, behavioral assays, TH intensity, and CHOP intensity analysis were as follows: nine female saline mice, 11 female cytisine mice, 11 male saline mice, and eight male cytisine mice (power = 0.9, effect size = 0.25, α = 0.05). For behavioral and in vitro experiments, sample size was determined using a power and sample size calculator in OriginLab 2019. Sample sizes were estimated using a power of 0.9, alpha value of 0.05, and an effect size of 0.25. For determining sample sizes, mean values and standard deviations were extracted from exemplar experiments that included saline versus cytisine-treated females and control versus 6-OHDA-treated DA neurons. Using the sample size calculator, we determined that in vivo and in vitro experiments required a minimum of eight mice and 40 cells per experimental group, respectively.

For behavioral assays and weight measurements, a two-way repeated measures analysis of variance (two-way ANOVA) was used to examine interactions between two factors that included treatment (saline versus cytisine) and time. A two-way repeated measures ANOVA was also used to examine the effects of sex on rotational behavior, with the two factors being sex and time. In both cases, the day of testing served as a repeated measure. Data were considered statistically significant when p < .05.

Data for DA cell cultures were separately obtained from two to three independent DA cultures per experiment. DA cells under each treatment condition were imaged across two to three independent DA cultures, pooled together and are presented in Figures 4-7. For statistical analysis of DA culture experiments, all datasets were first tested for normality using the Shapiro–Wilk test. A t-test was used for normally distributed data, whereas Mann–Whitney or Wilcoxon signed rank tests were used for unpaired and paired non-normal data, respectively. Differences across conditions were considered statistically significant when p < .05. Exact sample sizes used for each experiment are stated in figure legends.

We used bootstrap analyses to confirm the reproducibility of differences in fluorescence intensities for ATF6, XBP1, and CHOP across experimental conditions. Bootstrap is an established method for data with high variability or low sample size which uses iterative resampling with replacement to estimate the true distribution of a test statistic (Dwivedi et al., 2017; Maris, 2012; Wedenberg, 2013). For bootstrap analysis, the percent nuclear translocation of each of ATF6, XBP1, and CHOP in each of the experimental conditions was iteratively resampled with replacement (10,000 iterations). We tested the null hypothesis that the distribution of percent nuclear translocation for each stress marker is not different between control and experimental conditions. For each ER stress marker, p values were derived from the proportion of iterations in each experimental condition that exceed the mean values of the bootstrapped control condition. The null hypothesis was rejected if p was <.05.

3.1 In vivo assessment of neuroprotection by cytisine in mice with 6-OHDA-induced parkinsonism

In order to assess if low-dose cytisine attenuates PD-related behavioral deficits, we used a mouse model of parkinsonism with unilateral loss of SNc DA neurons, induced by a unilateral injection of 6-OHDA into the dorsolateral striatum (DLS). As shown in Figure 1a, starting at 7 days prior to striatal 6-OHDA injection (day −7), mice were injected i.p. with either 0.9% saline (vehicle) or a low, 0.2 mg/kg dose of cytisine on alternate days. 6-OHDA was stereotaxically injected into the DLS on day 0 and alternate day i.p. injections were continued until the mice were killed on day 21. Behavioral assays were conducted prior to cytisine and 6-OHDA treatment (day −7), and on days 7, 14, and 21 post-treatment. On day 21, the mice were perfused and 40 µm midbrain sections were obtained to quantify tyrosine hydroxylase (TH) content in the midbrain.

We first analyzed the effect of alternate day 0.2 mg/kg i.p. cytisine injections on the weight of female and male mice. No differences were observed between the weight of saline or cytisine-treated female and male mice on any day prior to or after 6-OHDA exposure. Injection of 6-OHDA into the DLS caused a 5%–10% weight loss in saline and cytisine-treated females and males, with no observable differences in the rate of weight gain between groups of female or male mice, and no statistically significant interaction between time and treatment as the two factors tested (females: F_(3,54) = 1.496; p = .226; males: F_(3,51) = 0.226, p = .878, two-way repeated measures ANOVA) (Figure 1b,c).

3.2 Cytisine attenuates apomorphine-induced rotational behavior in female mice

Intraperitoneal (i.p.) injection of apomorphine in mice with unilateral SNc lesions will increase rotations in a direction contralateral to the lesioned side. Rotation in mice occurs because of the excessive activation of hypersensitive dopamine 1 receptors in striatal medium spiny neurons (MSNs) on the lesioned side (Grealish et al., 2010). Therefore, the number of apomorphine-induced rotations can be used a quantitative measure of neuroprotection of SNc DA neurons in the 6-OHDA lesioned side. Based on this rationale, we assessed the extent of cytisine-induced neuroprotection in 6-OHDA lesioned female and male mice by quantifying the number of contralateral rotations following the systemic administration of apomorphine. All groups of mice were injected with 0.5 mg/kg apomorphine i.p., and the number of contralateral rotations were recorded for 15 min immediately after apomorphine injection (Figure 2a).

We first quantified contralateral rotations in saline-treated female and male mice. Compared to baseline rotations in mice prior to 6-OHDA lesioning, all 6-OHDA lesioned mice showed tight contralateral rotations following apomorphine injections (Figure 2a and Movies S1, S2). Rotations progressively increased from 80 ± 15 on day 7 to 118 ± 24 on day 21 in females and 40 ± 6 on day 7 to 80 ± 12 on day 21 in males (Figure 2b). Interestingly, saline-treated females showed ~twofold greater rotations when compared with saline-treated males on all days after 6-OHDA, and a significant interaction between time and sex (F_(3,54) = 4.141; p = .01, two-way repeated measures ANOVA).

Having verified the model for apomorphine-induced rotations in mice, we assessed the effect of cytisine treatment on contralateral rotations in 6-OHDA-treated females and males. Interestingly, cytisine-treated females showed twofold fewer rotations than saline controls on days 7, 14, and 21 after 6-OHDA (Figure 2c), and significant interaction between time and treatment (F_(3,54) = 3.834; p = .015, two-way repeated measures ANOVA). In contrast, cytisine-treated males rotated to a similar extent as saline controls (Figure 2c), and no interaction between time and treatment (F_(3,48) = 0.815; p = .492, two-way repeated measures ANOVA). These data demonstrate that cytisine attenuates rotational behavior only in female mice at concentrations that cannot activate nAChRs.

3.3 Cytisine attenuates gait pattern only in female mice

We analyzed gait patterns for all 6-OHDA-injected mice that were subjected to rotational assays. Gait was manually determined, counting normal versus abnormal patterns, during the ladder rung walking test (Figure 2d). At baseline (day −7), all mice showed normal gait patterns 100% of the time. However, following 6-OHDA treatment, saline-treated female and male mice showed a significant 40 to 50% decline in normal gait pattern (day −7 to day 7; females: p = .01; males: p = .002, Wilcoxon Signed Rank test) (Figure 2e and Movies S3, S4), indicating that unilateral 6-OHDA lesioning causes a significant deficit in the gait of both female and male mice.

Compared to saline controls, cytisine-treated females showed a significant 30% increase in normal gait pattern on day 21 after 6-OHDA (p = .03, Mann–Whitney test) (Figure 2e). In contrast, cytisine-treated male mice showed no improvement in gait pattern on days 7, 14, or 21 (Figure 2e). These data show that cytisine significantly attenuates gait abnormalities in 6-OHDA-treated female, but not in male mice.

3.4 Cytisine reduces foot slips only in female mice

As a third behavioral measure, we quantified foot slips in all mice during the ladder rung walking test (Figure 2f). We found that cytisine-injected females displayed ~50% fewer foot slips than saline controls on days 7, 14, and 21 following 6-OHDA lesioning and a significant interaction between time and treatment (F_(1,18) = 5.39; p = .032, two-way repeated measures ANOVA) (Figure 2g). In contrast, there was no effect of cytisine on foot slips in male mice (Figure 2g), and no significant interaction between time and treatment (F_(1,17) = 0.027; p = .871, two-way repeated measures ANOVA).

In summary, using three independent behavioral assays, we demonstrate that chronic systemic exposure to low-dose cytisine significantly reduces PD-related motor deficits in female, but not in male parkinsonian mice.

3.5 Cytisine reduces the loss of substantia nigra pars compacta (SNc) dopaminergic (DA) neurons in 6-OHDA-treated female mice

Since cytisine attenuated PD-related behaviors in female mice, we asked if cytisine exerts neuroprotection in the SNc of female mice. Midbrain sections from all mice subjected to behavioral assays were stained for tyrosine hydroxylase (TH) and montages were acquired from six midbrain sections per mouse (Figure 3a).

To assess the extent to which unilateral 6-OHDA injections induce SNc DA neuron loss in saline-treated female and male mice, we measured TH fluorescence intensity in the SNc from lesioned and unlesioned sides of midbrain sections. When compared with the unlesioned side, both female and male saline-treated mice showed an ~85% reduction in SNc TH intensity on the lesioned side (for females, integrated TH intensities in unlesioned SNc: 7.7 × 10⁶ ± 1.3 × 10⁶ arbitrary units (A.U.) and in lesioned SNc: 1.1 × 10⁶ ± 0.27 × 10⁶ A.U., p = 4.94 × 10^–10; for males, integrated TH intensities in unlesioned SNc: 9.7 × 10⁶ ± 1.46 × 10⁶ A.U. and in lesioned SNc: 1.6 × 10⁶ ± 0.36 × 10⁶ A.U., p = 3.64 × 10^–11, Mann–Whitney test) (Figure 3a,b). No significant sex differences were observed in total SNc TH content within either the unlesioned or lesioned side of saline-treated female and male mice (unlesioned: p = .36; lesioned: p = .61, Mann–Whitney test) (Figure 3b).

Next, we assessed if cytisine exerts a neuroprotective effect on SNc DA neurons in female mice. For each midbrain section, TH intensity ratios of the lesioned and unlesioned SNc were obtained in cytisine- and saline-treated female and male mice. The use of TH intensity ratios between lesioned and unlesioned SNc, rather than mean TH intensity in the lesioned SNc reduces variability that could occur between individual midbrain sections as a result of immunostaining procedure artifacts. When compared with saline-treated female mice, TH intensity ratios in the SNc were ~50% larger in cytisine-treated female mice, whereas cytisine- and saline-injected male mice showed no difference in TH ratios. The robust increase in SNc TH intensity ratios in cytisine-treated female mice was statistically significant regardless of whether mean TH intensity ratios were compared between individual groups of mice using an average TH intensity ratio obtained from multiple SNc midbrain sections for each mouse (n = 6 SNc midbrain sections per mouse; females: p = .05; males: p = .866, Mann–Whitney test) (Figure 3c), or when individual TH intensity ratios of the midbrain SNc were obtained for each individual section and then pooled for groups of female and male mice (females: p = 5.34 × 10⁻⁵; males: p = .562, Mann–Whitney test) (Figure 3d). These data show that cytisine exerts a neuroprotective effect in SNc DA neurons of 6-OHDA-treated female, but not male parkinsonian mice.

3.6 Cytisine treatment causes a pathological increase in the nuclear translocation CHOP only in 6-OHDA-treated male mice

Previous studies have shown that the degeneration of DA neurons by 6-OHDA requires CHOP expression (Aime et al., 2020; Silva et al., 2005). Therefore, we assessed the extent to which 6-OHDA causes the nuclear translocation of CHOP in lesioned SNc DA neurons of cytisine- and saline-treated female and male mice. DA neurons from the lesioned SNc were immunostained for CHOP and the extent of CHOP translocation into the nucleus of SNc DA neurons was assessed across all treatment groups in female and male mice (Figure 3e). We found that the percent nuclear translocation of CHOP in lesioned SNc DA neurons was significantly lower in saline-treated males when compared with saline-treated females (Figure 3f) (n = 60 female and 65 male cells; p = 3.02 × 10^–14, Mann–Whitney test). Furthermore, when compared with saline-treated males, cytisine-treated males showed a significant threefold increase in the percent nuclear translocation of CHOP (Figure 3f) (n = 65 saline and 70 cytisine cells; p = 2.49 × 10^–11, Mann–Whitney test). In contrast, no significant differences were observed between saline- and cytisine-treated females for nuclear CHOP translocation in lesioned SNc DA neurons (Figure 3f) (n = 60 saline and 69 cytisine cells; p = .174, Mann–Whitney test). Together, these data show a clear sex difference in the ability of the key pro-apoptotic ER stress molecule, CHOP to translocate into nuclei of SNc DA neurons.

3.7 Exposure to 6-OHDA causes cleavage and activation of pro-apoptotic caspase-3 in cultured primary mouse DA neurons

Based on the observation that cytisine causes a robust increase in nuclear CHOP translocation in SNc DA neurons of male but not female mice, we sought to determine if estrogen regulates CHOP activation in DA neurons. To do this, we generated primary cultures of mouse DA neurons, and first assessed if exposure of primary cultured mouse DA neurons to 6-OHDA is sufficient to initiate the apoptosis of cultured TH⁺ DA neurons. Because caspase-3 cleavage and activation is a known downstream mechanism for CHOP-mediated apoptosis (Quick & Faison, 2012; Teske et al., 2013; Wang et al., 2014), we specifically assessed the extent to which 6-OHDA induces caspase-3 cleavage in cultured midbrain DA neurons.

Primary midbrain neurons were cultured with midbrain astrocytes using previously published methods (Bancroft & Srinivasan, 2020; Henley et al., 2017; Srinivasan, Henley, et al., 2016). Our long-term 4-week-old primary mouse midbrain neuron-astrocyte co-cultures contain mature TH⁺ and TH^- neurons on a monolayer of S100B⁺ mouse midbrain astrocytes (Figure 4a) (Bancroft & Srinivasan, 2020; Henley et al., 2017; Srinivasan, Henley, et al., 2016). Four-week-old midbrain cultures were treated with 400 μM 6-OHDA for 48 hr, and immunostained for caspase-3 using an antibody specific to the cleaved form of caspase-3 (Figure 4c). In parallel experiments, the cleaved caspase-3 antibody was validated in midbrain cultures treated with 10 μM doxorubicin, which is known to cleave and activate caspase-3 (Ueno et al., 2006; Wei et al., 2015) (Figure 4b). 10 μM doxorubicin caused a significant and robust increase in immunostained cleaved caspase-3 in midbrain cultures, thus validating the antibody (p = 6.67 × 10⁻⁶, Mann–Whitney test) (Figure 4b). Using this validated cleaved caspase-3 antibody, we found that 48 hr exposure to 400 μM 6-OHDA caused a significant increase in cleaved caspase-3 expression in TH⁺ neurons (p = 4.03 × 10⁻⁵, Mann–Whitney test) (Figure 4c), indicating that 48 hr exposure to 400 μM 6-OHDA is sufficient to initiate apoptosis in primary cultured DA neurons.

3.8 17-β-estradiol alone inhibits CHOP expression in mouse DA neurons

Having established that 6-OHDA initiates apoptosis of cultured primary mouse DA neurons, we assessed the effect of cytisine and the major estrogen metabolite, 17-β-estradiol on CHOP in cultured primary mouse DA neurons exposed to 6-OHDA.

Primary mouse midbrain cultures were treated with either no drug (control), 6-OHDA (400 μM, 48 hr), 6-OHDA (400 μM, 48 hr) + cytisine (200 nM, 2 weeks), or 6-OHDA (400 μM) + cytisine (200 nM) + 17-β-estradiol (10 nM, 2 weeks) and for each treatment condition, CHOP fluorescence was quantified in either the whole cell or in the nucleus of TH⁺ DA neurons (Figure 5a,b). Population data from four independent midbrain cultures and ~ 100 DA neurons per condition showed that when compared with untreated control neurons, 6-OHDA significantly increased mean CHOP fluorescence across the whole cell and in the nucleus (whole cell: p = 1.46 × 10^–5; nucleus: p = 8.23 × 10^–6; Mann–Whitney test), and pre-exposure of 6-OHDA-treated cells to cytisine failed to reduce CHOP expression in either compartment (whole cell: p = .84; nucleus: p = .007; Mann–Whitney test) (Figure 5b). On the other hand, co-treatment of 6-OHDA + cytisine-exposed cells with 17-β-estradiol significantly reduced CHOP fluorescence in the whole cell and in the nucleus of DA neurons (whole cell: p = 7.27 × 10^–7; nucleus: p = 5.07 × 10^–7; Mann–Whitney test) (Figure 5b). To determine if 17-β-estradiol alone could reduce CHOP fluorescence, DA cultures were treated with 10 nM of 17-β-estradiol for 2 weeks, and baseline CHOP fluorescence intensities in the control and 17-β-estradiol-treated conditions were measured. When compared with untreated control cultures, 17-β-estradiol alone caused a significant reduction in mean CHOP fluorescence across the whole cell (p = .003; Mann–Whitney test) (Figure 5c).

Based on these data, we conclude that 17-β-estradiol alone decreases CHOP expression to baseline levels in cultured DA neurons, and this occurs at nanomolar concentrations that are physiologically relevant to in vivo 17-β-estradiol concentrations.

3.9 Cytisine up-regulates Sec24D-containing ER exit sites (Sec24D-ERES) in primary cultured mouse DA neurons

The observation that 17-β-estradiol alone inhibits CHOP expression in DA neurons led us to assess the effect of cytisine on all the three key proteins, ATF6, XBP1, and CHOP that are known to play a central role in regulating each of the three arms of ER stress signaling.

Ligand-mediated chaperoning of nAChRs up-regulates Sec24D-ERES, which can reduce ER protein burden and mitigate pathological ER stress (Srinivasan, Henley, et al., 2016; Srinivasan et al., 2011, 2012). Since cytisine binds to nAChRs with picomolar affinity (Coe et al., 2005), and up-regulates α4β2* nAChRs (* denotes other unknown pentameric nAChR subunits) (Richards et al., 2011, 2012; Srinivasan et al., 2011), we first asked if nanomolar concentrations of cytisine can chaperone nAChRs and consequently, up-regulate Sec24D-ERES in cultured DA neurons.

To assess cytisine-induced Sec24D-ERES up-regulation, DA cultures were treated with 200 nM cytisine for 2 weeks and endogenous Sec24D-ERES sites were immunolabeled and quantified with confocal microscopy (Figure 6a). Compared to untreated control cultures, 2 weeks of cytisine exposure significantly increased the number, area, and intensity of Sec24D-ERES in TH⁺ DA neurons (p = .05 for number, p = .027 for area, p = 9.5 × 10^–4 for intensity; Mann–Whitney test) (Figure 6b). These data demonstrate that cytisine up-regulates Sec24D-ERES in DA neurons at doses that cannot activate surface nAChRs.

3.10 Cytisine attenuates the nuclear translocation of ATF6 and XBP1, but not CHOP in 6-OHDA-treated primary cultured mouse DA neurons

Four-week-old primary mouse DA neuron-astrocyte co-cultures were incubated with 400 µM 6-OHDA for 48 hr and the nuclear translocation of three key ER stress response proteins, ATF6, XBP1, and CHOP were quantified in untreated versus 6-OHDA-treated and 6-OHDA + cytisine-treated cultures. We found that 6-OHDA significantly increased nuclear translocation of ATF6, XBP1, and CHOP in cultured primary mouse TH⁺ neurons (ATF6: p = .002, XBP1: p = .06, CHOP: p = 1.6 × 10^–7; Mann–Whitney test) (Figure 7a,b). Co-exposure of DA cultures to cytisine (200 nM for 2 weeks) and 6-OHDA (400 μM for 48 hr) significantly decreased nuclear ATF6 and XBP1 translocation in TH⁺ DA neurons (ATF6: p = 1.87 × 10^–9, XBP1: p = .003; Mann–Whitney test), and caused a significant increase in nuclear CHOP fluorescence (p = .02, Mann–Whitney) (Figure 7b).

To account for variability in ER stress levels across independent primary DA cultures, we applied bootstrap analysis for all experimental data from Figure 7b. Ten thousand iterations of bootstrap re-sampling and replacement were performed using experimentally derived nuclear intensities of ATF6, XBP1, and CHOP. 6-OHDA showed a significant pathological increase in the nuclear translocation of ATF6, XBP1, and CHOP (ATF6: p < .0001, XBP1: p < .0001, CHOP: p < .001). Consistent with the experimental data, cytisine significantly reduced the 6-OHDA-mediated increases of nuclear ATF6 and XBP1 levels with no attenuation of 6-OHDA-induced nuclear CHOP translocation (ATF6: p = .03; XBP1: p < .0001; CHOP: p = .14; bootstrap analyses) (Figure 7c). Thus, chronic exposure to 200 nM cytisine attenuated only two of the three signaling arms of 6-OHDA-induced hyperactive ER stress (ATF6 and XBP1), without affecting 6-OHDA-induced CHOP activation in DA neurons.

Taken together, these results show that in cultured DA neurons, 17-β-estradiol inhibits CHOP expression, whereas cytisine reduces the nuclear translocation of ATF6 and XBP1. Thus, the combination of 17-β-estradiol and cytisine attenuates all three arms (ATF6, XBP1 and CHOP) of the ER stress signaling pathway in DA neurons.